Methods of oxygenating tissues

ABSTRACT

The present invention provides a method of delivering an emulsion or suspension containing a supersaturated gas into a gas-depleted environment. The method generally comprises the steps of preparing an emulsion or suspension, exposing the emulsion or suspension to a gas at a pressure greater than 2 bar, and delivering the emulsion or suspension to a gas-depleted environment at ambient pressure.

This application is a Continuation of application Ser. No. 08/889,634now U.S. Pat. No. 5,922,305 filed Jul. 8, 1997, which is a Divisional ofapplication Ser. No. 08/730,517 now U.S. Pat. No. 5,834,519, filed Oct.11, 1996.

TECHNICAL FIELD

This invention relates to a method for preparing a gas-supersaturatedemulsion or suspension and delivering it from a high pressureenvironment to a gas-depleted site without the immediate onset ofcavitation or bubbling.

BACKGROUND ART

The maximum concentration of gas achievable in a liquid is governed byHenry's Law. The relatively low solubility at ambient pressure of manygases (for example, oxygen or nitrogen) within a liquid such as waterresults in a low concentration of the gas in the liquid when these aremixed. There are, however, many applications where it would beadvantageous to employ a gas in a liquid mixture where the concentrationof the gas within the liquid greatly exceeds its solubility at ambientpressure.

High-pressure compression of a liquid within a liquid emulsion or solidwithin a liquid suspension can be used to achieve a higher dissolved gasconcentration, but disturbance of this gas supersaturated liquid throughejection into a 1 bar environment from the high pressure reservoir willgenerally result in cavitation inception at or near the exit port. Therapid evolution of bubbles produced at the exit port vents much of thegas from the liquid, so that the high degree of gas concentration withinthe liquid is considerably reduced at the ambient pressures outside thehigh pressure vessel. Additionally, the presence of bubbles in theeffluent generates turbulence and impedes the flow of the effluentbeyond the exit port.

A wide variety of applications would benefit from ejection of agas-supersaturated fluid from a high pressure reservoir into an ambientpressure environment in a manner which does not involve cavitationinception at or near the exit port. For example, organic material andplant waste streams—e.g., paper mills and chemical plants—often requirean increase in dissolved oxygen content before these streams can besafely discharged into a body of water. U.S. Pat. No. 4,965,022recognizes that a similar need may also occur at municipal wastetreatment plants and that fish farms require increased dissolved oxygenlevels to satisfy the needs of high density aquaculture. Otherapplications are disclosed in U.S. Pat. No. 5,261,875.

There are many prior art references which disclose methods of enrichingthe oxygen content of water. For example, U.S. Pat. No. 4,664,680discloses several conventional types of apparatus that can be used forcontinuously contacting liquid and oxygen-containing gas streams toeffect oxygen absorption within the liquid. Specifically, pressurizableconfined flow passageways are used to avoid premature liberation of thedissolved oxygen before it is incorporated within the fluid. Otheroxygen saturation devices are disclosed in U.S. Pat. Nos. 4,874,509 and4,973,558. However, these techniques leave unsolved the problem of howto eject the gas-enriched fluid solutions from a high pressure reservoirinto a lower pressure environment without the formation of bubbles inthe effluent at or near the exit port.

In a previous application Ser. No. 08/581,019, filed Jan. 3, 1996, Idescribe a method for ejection of gas-supersaturated liquids from a highpressure to a low pressure environment without cavitation, consisting ofextrusion of the fluid through capillary channels and compression toremove cavitation nuclei along the inner surface of the channels.Hydrostatic compression at pressures between 0.5 kbar and 1.0 kbarrapidly removes cavitation nuclei and bubbles from the liquid. When agas source is used to both pressurize the liquid and achieve a desiredconcentration of a relatively insoluble gas in the liquid, it isgenerally necessary to maintain the gas pressure in the 10 bar to 150bar range.

The complete absence of cavitation inception in water saturated withoxygen at high concentrations permits its in vivo infusion into eithervenous or arterial blood for the purpose of increasing the oxygenconcentration of the blood while avoiding the formation of bubbles whichtend to occlude capillaries.

In contrast to this capillary channel technique, the present inventiondispenses with the necessity of compressing fluids within capillarychannels, relying instead on use of gas-supersaturated emulsions andsuspensions.

SUMMARY OF THE INVENTION

A method is described for the use of emulsions or suspensions totransport a gas-supersaturated liquid from a high pressure reservoir toa relatively low pressure environment (including ambient pressure),without immediate cavitation inception.

If a liquid that has a relatively high gas solubility (also known as theinternal phase) is suspended in fine droplets within another immiscibleliquid or semi-solid having a relatively low gas solubility (known asthe carrier or external phase) a high level of supersaturation of thegas can be achieved in the resulting emulsion upon its release to agas-depleted environment at ambient pressure. Likewise, solid particlescan be suspended within a liquid carrier to form a suspension with thesame properties (unless otherwise indicated, the descriptions for liquidin liquid emulsions are true for solid in liquid suspensions as well).The primary gases of interest for the formation of gas supersaturatedemulsions are oxygen, nitrogen, and carbon dioxide.

The small size of the droplets or particles in conjunction with exposureto a transient high hydrostatic pressure confers stability to thedroplets or particles in a manner similar to that provided by smalldiameter capillary tubes. Generally, the fine droplets are between about0.1 micron and about 10 microns in diameter. Thus, after release of theemulsion to an ambient pressure environment, the gas that is dissolvedat high levels of supersaturation will not form bubbles, despite arelatively high concentration of the gas within the droplets orparticles.

The carrier of the droplets or particles is stable at high gas partialpressures because of the relatively low gas solubility of the carrier aswell as the absence of gas nuclei after hydrostatic compression. A lowgas diffusion coefficient in the carrier results in a slow, delayedrelease of the gas both from the droplets or particles to the carrier aswell as from the emulsion to the gas-depleted environment. Despite thisslow release of gas from the emulsion and the relatively lowconcentration of gas in the carrier, the high partial pressure of gas inthe emulsion creates a high driving pressure gradient between theemulsion and gas-poor surfaces.

As a result of the lack of cavitation inception at or near the exitport, a stream of the gas-supersaturated emulsion can be used to rapidlyand efficiently enrich a gas-deprived site such as a liquid byconvection of the emulsion to the gas-deprived site. Enrichment of agas-deprived liquid with gas by diffusion from the gas phase to theliquid is, by contrast, an extremely slow process.

The lack of bubbles in the effluent additionally permits unimpededejection into the gas-depleted site. When the gas-supersaturatedemulsion is ejected in an air environment, the lack of cavitationinception at or near the exit port allows the effluent to behave as ifit were not supersaturated with gas. That is, the ejected stream remainsintact rather than disintegrating into a diffuse spray near the exitport due to the rapid growth of gas nuclei.

The basic steps for forming the gas-supersaturated emulsion (the samemethod applies to the formation of a gas-supersaturated suspension) are:preparing the emulsion; exposing the emulsion to a gas at a pressuregreater than 2 bar; and delivering the emulsion to a gas-depletedenvironment at ambient pressure. Typically, the emulsion is exposed tothe gas (the primary gas of interest is oxygen) at a pressure of betweenabout 5 bar and about 20 bar. The emulsion could be rapidly mixed (atabout 1600 rpm, for instance) for several hours during its exposure tothe gas at partial pressures between 100 psi and 1500 psi. Additionally,the emulsion could be delivered to a high pressure hydrostatic pump inorder to further increase the partial pressure of the gas.

The emulsion is extruded at the output of a pressurizable vessel througha tube, which delivers the emulsion to the outside environment atbetween about 0.1 and about 10 ml per minute.

This type of emulsion can be used to efficiently deliver oxygen to theskin, to wounds, or to other environments. In a biological context, thehigh level of oxygen achieved in such tissues by contact of the emulsionwith the tissues should be helpful in a variety of ways, such ascollagen synthesis, inhibition of anaerobic bacterial growth, andpromotion of aerobic metabolism. A supersaturated oxygen emulsion canalso be used to oxygenate blood for a variety of medical applications.The emulsion is injected directly into the bloodstream, therebyincreasing oxygen delivery to the blood.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Forming the Emulsion

To form an emulsion, a liquid which will be suspended as droplets withina carrier as well as the carrier must be chosen. The carrier for theemulsion includes any liquid or semi-solid having a relatively lowdiffusion rate of the gas to be dissolved. The same techniques apply tothe formation of a suspension of particles within a liquid carrier.

In general, liquids characterized by high viscosity and low gassolubility are the most effective carriers, since these properties tendto increase the liquid's resistance to bubble formation. Ideally, theviscosity of the carrier should be in the 1 centipoise to 10 centipoiserange. Examples of commonly used carriers include glycerin, gels such ashydrogel, vaseline, paraffin, and waxes.

Gelatins also make effective carriers. For example, 5-10 wt % gelatinswere cross-linked in glutaraldehyde to render them insoluble in water,exposed to oxygen at 10-20 bar, and subsequently compressed at highhydrostatic pressures (e.g., 0.5 to 10 kbar) for about an hour. Uponrelease to hydrostatic pressure of 1 bar, it was noted that no bubblesformed in the gelatin, and a surface pO₂>2000 mm Hg was maintained forperiods of at least 20-30 minutes.

In contrast to the carrier, the liquid droplets or solid particles whichare to be suspended in the carrier must have a high gas solubility. Forexample, perfluorochemical (PFC) droplets can be suspended withingelatin by adding a hot gelatin solution to a PFC droplet concentrate,mixing briefly, and cooling to affect solidification. With sufficientlyhigh oxygen pressure, the PFC droplets will absorb a high concentrationof oxygen and maintain stability at 1 bar by virtue of their small size.Likewise, the gelatin will provide a slow rate of diffusion of oxygenfrom the particles and through the gelatin to the oxygen-poor site. Onesuch environment that this suspension could be applied to is biologictissue.

In addition to PFC droplet, other droplet materials that could be usedto provide a stable depot of concentrated oxygen include lipids,liposomes, and oils (the class of oils including mineral, coconut, andvegetable oils), most of which have a high solubility of oxygen relativeto that of water.

Solid particles useful in preparing suspensions of the present inventionare composed of polymers. These polymers have been found to absorb gasessuch as oxygen under high pressure conditions and to release the gaseswithout bubble formation upon exposure to ambient pressure. Thepreferred polymers include polyacrylamide (in either its unhydrated orhydrated form), polypropylene, polycarbonate, polyethylene, polylacticacid, polyglycolic acid, polycaprolactone, polyethylene glycol,polystyrene, polysorbate, polymethyl methacrylate and co-polymersthereof. Preferably, the size of the solid particles are within therange of 0.1 to 10 micron.

Any particle or droplet could also be micro or nano-encapsulated with asemi-permeable surface coating that further controls the rate ofdiffusion from the particle or droplet to the carrier. Encapsulation canbe achieved through well-known techniques such as coacervation or vapordeposition.

In order to form an emulsion, one can obtain a commercially availableemulsion comprising a desired liquid suspended in water. Aftercentrifuging this emulsion and decanting the supernatant, the desireddroplets can be resuspended in a carrier of choice. Likewise, in forminga suspension, one can centrifuge a suspension of particles, decant thesupernatant, and resuspend the particles in another carrier of choice.

Preferred Embodiment

The following example is provided to illustrate the above principles.Glycerin was chosen as a carrier because of its low oxygen solubility(0.008 cc O₂/g/atm.), relatively high viscosity, and low rate of oxygendiffusion. Moreover, it is a biocompatible liquid, thereby allowingapplication to the skin or to wounds. Perfluorochemical (PFC) particleswere chosen to be suspended in the carrier due to their high oxygensolubility (0.5 cc O₂/g/atm.), their inherent ability to form into smallparticles (typically equal to or less than 0.5 μm), and theirbiocompatibility.

In order to prepare the PFC/glycerin suspension, previously preparedcommercially available PFC/aqueous suspensions were centrifuged. The PFCparticles at the bottom of the centrifuge tubes were resuspended inglycerin after decanting the supernatant.

The PFC/glycerin suspension (200 ml) was placed in a 300 ml capacityParr reactor vessel, and the suspension was exposed to oxygen at partialpressures as high as 500 to 1500 psi during rapid mixing (at about 1600rpm) with an impeller stirrer. High oxygen partial pressures wererequired to drive the oxygen into the suspension over a period of manyhours because of the slow rate of diffusion of oxygen through theglycerin.

Despite the high oxygen partial pressures, the oxygen partial pressuredeveloped in the suspension after the above treatment and overnightexposure to oxygen at 300 psi (without stirring) was estimated to beapproximately 10 atm. After delivery of the suspension to a Haskel highpressure hydrostatic pump at 1000 psi oxygen partial pressure, thehydrostatic pressure increased to 12,000 psi. At the output of the pump,a 0.009 inch i.d. stainless steel tube—about 100 cm long—was used todeliver the suspension to the outside ambient environment at a flow rateof about 0.2 ml/min.

No bubbles formed in the suspension after extrusion of the suspensioninto a glass beaker, plastic test tube, or skin (including manualspreading of the suspension on the skin of a hand).

However, the pO₂ in the suspension was approximately 10 times higherthan that noted in glycerin that had been exposed only to air, asdetermined with a polarographic type membrane pO₂ electrode(manufactured by Diamond General, Ann Arbor). Aliquots of 1 ml of thesuspension were in communication with a column of mercury for measuringvolume changes at 1 bar as well as in contact with a prototype titaniumprobe (distal end of which contacted the upper portion of the liquidsample). The probe was seated within the pipette by means of a collarthat had been built into the device at its node and glued into theinside of a tube that communicated with the pipette. The probe, drivenby a 1500 watt amplifier (manufactured by Sonics and Materials, Inc.),was used to degas the liquid sample during 1 minute periods ofsonication.

It was found that the suspension contained approximately 1 ml O₂/g.Since the suspension ordinarily contains about 0.1 ml O₂/g/bar in water,and the percent volume of PFC in glycerin is similar to that in water,the partial pressure of the gas must have been about 10 bars.

In order to determine how long the suspension retained the high oxygenconcentration, the measurement of the oxygen concentration was repeatedat 5, 10, 20, and 30 minutes after delivery of the suspension into a 50ml beaker. Over the first 10 minutes, only 30% of the oxygen was lostfrom the suspension; however, by 30 minutes, most of the oxygen haddiffused out. Thus, it is apparent that the diffusion of oxygen from thesuspension is quite slow, partly as a result of the relativelyimpermeable nature of the carrier.

Dispensing the emulsion

A simple dispenser for the oxygen-rich cream emulsion can consist of asyringe type design, with the barrel driven by manual rotation of apiston that advances as a screw on threads, similar to the operation ofcommercially available “indeflators” used to pressurize high pressureballoons (as high as 300 psi) on angioplasty catheters.

Manual compression to at least 300 psi is easily achievable, and a valveat the distal end of the syringe would allow the cream to be squeezedfrom the syringe in a controlled manner. After dispensing a desiredamount of cream, the stopcock would be closed and additional pressureapplied to maintain a hydrostatic pressure that equals or exceeds thedissolved gas partial pressure. The syringe would be fabricated frommaterials that are impermeable to oxygen.

It should be noted that there are a wide variety of geometries whichcould be employed at or near the exit port(s) which would permit theejection of the cavitation-free, gas-supersaturated emulsion into a 1bar environment from a high pressure reservoir. For example, I havefound that a 50 micron diameter square borosilicate glass tubing worksas effectively as both a round glass tubing and a round stainless steeltubing of similar diameter for this purpose. A rectangular or slit-likegeometry characterizing the delivery channels would also be expected tobe effective.

Although the present invention has been described and illustrated indetail, it is clearly understood that the same is by way of illustrationand example only and is not to be taken by way of limitation, the spiritand scope of the present invention being limited only by the terms ofthe appended claims.

All references cited in the present specification are incorporated byreference.

What is claimed is:
 1. A method of treating a biologic tissue woundcomprising the act of applying to the wound an oxygen-supersaturatedemulsion to treat the tissue by increasing oxygen delivery to the wound.2. A method of supplying oxygen to skin to promote collagen synthesis,comprising the act of applying to the skin an oxygen-supersaturatedemulsion to increase oxygen delivery to the skin.
 3. A method ofoxygenating tissue, comprising applying to the tissue anoxygen-supersaturated emulsion to cause oxygen delivery from theemulsion to the tissue.
 4. A method of supplying oxygen to tissue,comprising providing proximate the tissue an oxygen-supersaturatedemulsion to cause oxygen diffusion from the emulsion to the tissue.
 5. Amethod of treating a wound, comprising applying an oxygen-supersaturatedemulsion to the wound, thereby treating the wound by delivering oxygento the wound.
 6. A method of supplying oxygen to skin, comprisingapplying an oxygen-supersaturated emulsion to the skin to cause thetransfer of oxygen from the emulsion to the skin.
 7. A method ofsupplying oxygen for delivery to a wound, comprising providing anemulsion for application to the wound as an oxygen-supersaturatedemulsion thereby delivering oxygen to the wound.
 8. A method ofsupplying oxygen for delivery to skin, comprising providing an emulsionto be applied to the skin as an oxygen-supersaturated emulsion, therebydelivering oxygen to the skin.
 9. A method of supplying oxygen fordelivery to a wound, comprising providing a container including anoxygenated emulsion which delivers oxygen to the wound upon applicationto the wound as an oxygen-supersaturated emulsion.
 10. A method ofsupplying oxygen for delivery to tissue, comprising providing within acontainer an oxygenated emulsion which delivers oxygen to the tissueupon application to the tissue as an oxygen-supersaturated emulsion. 11.A method of supplying oxygen for delivery to tissue, comprisingproviding a container having an oxygenated emulsion which deliversoxygen to the tissue upon application to the tissue as anoxygen-supersaturated emulsion.
 12. A method of supplying oxygen fordelivery to tissue, comprising introducing into a container anoxygenated emulsion which delivers oxygen to the tissue upon applicationto the tissue as an oxygen-supersaturated emulsion.
 13. A method ofsupplying oxygen for transfer to tissue, comprising providing adispenser containing an oxygenated emulsion which delivers oxygen to thetissue upon application to the tissue as an oxygen-supersaturatedemulsion.
 14. A method of supplying oxygen for transfer to tissue,comprising providing within a container an oxygenated emulsion whichdelivers oxygen to the tissue upon application to the tissue as anoxygen-supersaturated emulsion.
 15. A method of supplying oxygen fortransfer to tissue, comprising introducing into a container anoxygen-supersaturated emulsion which delivers oxygen to the tissue uponapplication to the tissue.
 16. A method for treating a biologic tissuecomprising the acts of: (a) providing a dispenser containing anoxygenated emulsion under at least 2 bars of pressure; and (b)dispensing the emulsion from the container onto the tissue to form anoxygen supersaturated emulsion which treats the tissue by increasingoxygen delivery to the tissue.
 17. A method of supplying oxygen tobiologic tissue comprising the act of: permeating an oxygensupersaturated emulsion into the tissue to deliver oxygen to the tissueover a period of time.